Project description:High resolution mass spectrometry-based proteomics generates large amounts of data, even in the standard liquid chromatography (LC) – tandem mass spectrometry configuration. Adding an ion mobility dimension vastly increases the acquired data volume, challenging both analytical processing pipelines and especially data exploration by scientists. This has necessitated data aggregation, effectively discarding much of the information present in these rich data sets. Taking trapped ion mobility spectrometry (TIMS) on the quadrupole time of flight platform (Q-TOF) as an example, we developed an efficient indexing scheme that represents all data points as detector arrival times on scales of seconds (LC), milliseconds (TIMS) and microseconds (TOF). In our open source AlphaTims package, data are indexed, accessed and visualized by a combination of tools of the scientific Python ecosystem. We interpret unprocessed data as a sparse 4D matrix and use just in time compilation to machine code with Numba, accelerating our computational procedures by several orders of magnitude while keeping to familiar indexing and slicing notations. For samples with more than six billion detector events a modern laptop can load and index raw data in about a minute. Loading is even faster when AlphaTims has already saved indexed data in a HDF5 file, a portable scientific standard used in extremely large-scale data acquisition. Subsequently, data accession along any dimension and interactive visualization happen in milliseconds. We have found AlphaTims to be a key enabling tool to explore high dimensional LC-TIMS-Q-TOF data and have made it freely available as an open-source Python package with a stand-alone graphical user interface at https://github.com/MannLabs/alphatims or as part of the AlphaPept framework.
Project description:MIADB: a cumulative collection of 172 tandem mass spectrometry (MS/MS) of a vast array of monoterpene indole alkaloids. Samples were analyzed using an Agilent LC-MS system composed of an Agilent 1260 Infinity HPLC coupled to an Agilent 6530 ESI-Q-TOF-MS operating in positive mode. A Sunfire analytical C18 column (150 × 2.1 mm; i.d. 3.5 μm, Waters) was used, with a flow rate of 250 μL/min and a linear gradient from 5% B (A: H2O + 0.1% formic acid, B: MeOH) to 100% B over 30 min. ESI conditions were set with the capillary temperature at 320 °C, source voltage at 3.5 kV, and a sheath gas flow rate of 10 L/min. The divert valve was set to waste for the first 3 min. There were four scan events: positive MS, window from m/z 100−1200, then three data-dependent MS/MS scans of the first, second, and third most intense ions from the first scan event. MS/MS settings were three fixed collision energies (30, 50, and 70 eV), default charge of 1, minimum intensity of 5000 counts, and isolation width of m/z 2. In the positive-ion mode, purine C5H4N4 [M + H]+ ion (m/z 121.050873) and the hexakis(1H,1H,3H-tetrafluoropropoxy)-phosphazene C18H18F24N3O6P3 [M + H]+ ion (m/z 922.009 798) were used as internal lock masses. Full scans were acquired at a resolution of 11 000 (at m/z 922). A permanent MS/MS exclusion list criterion was set to prevent oversampling of the internal calibrant.
Project description:Data-independent acquisition (DIA) on ion mobility – TOF hybrid mass spectrometers enables deep proteome coverage and high data completeness in large-scale proteomics studies. For advanced acquisition schemes such as parallel accumulation serial fragmentation-based DIA (diaPASEF) stability of ion mobility (1/K0) over time is crucial for consistent data quality. We found that minor changes in environmental air pressure systematically affect vacuum pressure in the TIMS analyzer, causing ion mobility shifts. By comparing experimental ion mobilities with historical weather data, we attributed observed drifts to daily fluctuations in ground air pressure. These drifts negatively impact peptide quantification across consecutively acquired samples due to drift-dependent abundance changes and increased missing values for ions located at the boundaries of diaPASEF isolation windows, which cannot be corrected by post-processing. To address this, we applied an in-batch mobility autocalibration feature on a run-wise basis, leading to full elimination of ion mobility drifts.